The invention relates to the identification and cloning of polynucleotides encoding proteases.
Proteases are involved in a variety of cellular processes, such as tumor invasion, wound healing, tissue remodeling, infection, and inflammation. Previously unknown proteases whose substrate specificities are known can be used, for example, to design compounds which can affect these processes, including therapeutic compounds for conditions such as cancer, inflammation, rheumatoid arthritis and other autoimmune diseases, and AIDS.
Many methods are available in the art for detecting protease activity. For example, WO 97/08194 allegedly discloses a method of assaying for protease activity by measuring the fluorescence intensity of a fluorescent substrate. It would be advantageous to have a method in which detection of protease activity is linked to identification of a polynucleotide encoding the protease. Thus, there is a need in the art for convenient and rapid methods which can be used both for detection of protease activity and for identification of polynucleotides encoding the protease.
It is an object of the present invention to provide methods for identifying coding sequences for proteases which are expressed under a variety of conditions, including proteases which were previously unknown. This and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides a method of identifying a candidate polynucleotide molecule encoding a protease. A first plurality of target cells which contain candidate polynucleotides is incubated under conditions which permit packaging of RNA corresponding to the candidate polynucleotides into viral display packages. The candidate polynucleotides may or may not encode a protease. The viral display packages display chimeric envelope proteins which comprise (I) a substantially intact viral envelope protein which enhances fusion between a viral membrane and a target cell membrane, (ii) an inhibitory protein which prevents fusion between the substantially intact viral envelope protein and a target cell membrane, and (iii) a protease recognition site located between the substantially intact viral envelope protein and the inhibitory protein. In another embodiment, the viral display packages display recombinant envelope proteins, in which a protease recognition site has been substituted for a furin cleavage site located between a large glycoprotein subunit of the envelope protein and a transmembrane component of the envelope protein. Recombinant envelope proteins can be derived from viral envelope proteins such as a Moloney murine leukemia virus envelope protein, a 4070A envelope protein, or an influenza virus envelope protein.
A xe2x80x9cprotease recognition sitexe2x80x9d according to the invention is a contiguous sequence of amino acids connected by peptide bonds which are recognized by a protease. Recognition of this site by a protease results in cleavage (i.e., hydrolysis) of peptide bond by the protease. The site of hydrolysis may be coincident with the protease recognition site, that is, the protease recognition site may include one or more amino acids on either side of the peptide bond to be hydrolyzed which are recognized by the particular protease. The specific sequence of amino acids in the protease recognition site depends on the catalytic mechanism of the protease, which is defined by the nature of the functional group at the protease""s active site. Alternatively, the protease recognition site may be one, two, three, four or more amino acids distal at the amino or carboxy terminus, to the site of cleavage by the protease.
The viral display packages produced by the first plurality of target cells are contacted with a second plurality of target cells. Infection of a member of the second plurality of target cells by a viral display package occurs only if a protease produced in a member of the first plurality of target cells removes the inhibitory protein from the chimeric envelope protein or, if a recombinant envelope protein is displayed, only if a protease produced in a member of the first plurality of target cells cleaves at the protease recognition site between the large glycoprotein subunit and the transmembrane component of the recombinant envelope protein to activate the envelope protein""s fusion activity. In a preferred embodiment, at least a third plurality of target cells is infected with a viral display package. If desired, the viral display package comprises a transferable label. The first, second, and at least third pluralities of target cells can be present in the same tissue culture vessel or in different tissue culture vessels.
Target cells can comprise a retroviral packaging signal, a viral long terminal repeat, and polynucleotides which encode packaging defective Gag, Pol polypeptides and a chimeric envelope protein. If desired, the retroviral packaging signal, the viral long terminal repeat, and the polynucleotides which encode Gag and Pol polypeptides and the chimeric envelope protein can be encoded in the same polynucleotide molecule as a candidate polynucleotide. If desired, members of the first and second pluralities of target cells can be the same cell type, such as a retroviral packaging cell line.
Target cells may be treated with growth factors, activating proteases or other protease-modulating compounds to modulate the protease activity of the target cells.
As used herein, the term xe2x80x9ctreatment to modulate protease activityxe2x80x9d refers to a process or treatment that results in activation of a protease expressed in a zymogen or pro-enzyme form. This treatment may, for example, activate or even introduce a proteolytic enzyme required for cleavage of a zymogen form of a protease.
As used herein, the term xe2x80x9cgrowth factorxe2x80x9d refers to a polypeptide that alters protease activity in a cell through interaction of the polypeptide with a specific receptor expressed by that cell.
As used herein, the term xe2x80x9cactivating proteasexe2x80x9d refers to a proteolytic activity that, through cleavage of one or more polypeptides, modulates the activity of a cellular protease.
As used herein, the term xe2x80x9cprotease-modulating compoundxe2x80x9d refers to a compound that directly or indirectly activates a cellular protease.
In preferred embodiments, the chimeric envelope protein comprises a substantially intact retroviral envelope protein, preferably a murine leukemia virus envelope protein, such as a 4070A or Moloney murine leukemia virus envelope protein.
In one embodiment, the inhibitory protein binds to a receptor present on the outer cell membrane of the target cell. The inhibitory protein can be, for example, CD3 antigen, epidermal growth factor, stem cell factor, and an insulin-like growth factor I.
In another embodiment, a first inhibitory protein oligomerizes with at least second inhibitory protein, such as CD40 ligand or a leucine zipper polypeptide, for example GCN4, C/EBP, Fos, Jun, and c-myc.
Candidate polynucleotides can be obtained from a cell which is not known to express a protease, from a cell which is known to express a protease, or from a cell which is likely to express a protease, such as a tumor cell, a cell of a tissue which is inflamed, a cell of a tissue which is undergoing remodeling, a cell of a tissue which is involved in wound healing, or a cell comprising an infectious agent which expresses a protease. Optionally, candidate polynucleotides are synthetic polynucleotides.
Sequences which permit integration of candidate polynucleotides into the genome of a target cell, such as retroviral long terminal repeats, can be included in candidate polynucleotide molecules. Endoplasmic reticulum or Golgi retention/retrieval signals also can be included in candidate polynucleotide molecules.
Preferably, primers are included in the candidate polynucleotide molecules, for use in amplifying a candidate polynucleotide molecule which encodes a protease. Amplified candidate polynucleotide molecules can then be sequenced.
The protease recognition site can be a variation of a cleavage site of a known protease. Preferably, the variation of the known cleavage site is formed by modifying at least one amino acid of the known protease recognition site.
Thus, the present invention provides an innovative approach to the identification of protease coding sequences.